New & Noteworthy

A goldfish will swim faster through water than it will through gravy or Jell-O. And according to the results of a new study by Miermont and coworkers, it looks like the same may be true for proteins in a yeast cell.

Protein movement in an osmotically stressed cell is like a goldfish in Jell-O – mighty slow!

Now obviously the authors didn’t replace the insides of a yeast cell with gravy or Jell-O. Instead, they used severe osmotic stress to remove water from the cell, making the interior more viscous and the proteins more crowded.

The movement of a wide range of proteins within the cell slowed to a crawl in these shrunken cells. In fact, if the cell was subjected to enough stress, the proteins in the cell essentially stopped moving at all. This lack of movement wasn’t because the high osmotic stress had killed the yeast…they recovered just fine when put back into a more osmotically friendly environment.

The first case the authors looked at was the pathway that helps yeast respond to osmotic stress, the HOG pathway. Once a cell is subjected to osmotic stress, Hog1p is phosphorylated and translocated into the nucleus where it can then turn on the genes needed to respond to this environmental insult.

Miermont and coworkers traced the movement of Hog1p using a Hog1p-GFP fusion protein. At 1 M sorbitol, which subjects the cell to mild osmotic stress, this fusion protein was phosphorylated within two minutes and had reached the nucleus within five. There were already signs of cell shrinkage even under these mild conditions.

As sorbitol concentrations increased, the phosphorylation and nuclear localization took longer and longer to happen. At 1.8 M sorbitol, Hog1p-GFP didn’t reach maximum fluorescence in the nucleus until 55 minutes. At 2 M sorbitol and above, the cells were shrunken down to their minimal volume, which was 40% of their original size. Under those conditions, only 25% of cells had any nuclear fluorescence even after several hours.

The authors used fluorescence recovery after photobleaching (FRAP) to confirm that these effects were due to slowed protein movement. Basically they zapped a small portion of a cell to burn out the fluorescence of the Hog1p-GFP in that region and then timed how long it took fluorescing Hog1p-GFP from other parts of the cytoplasm to diffuse into that area. To prevent the complication of nuclear translocation in these experiments, they used a pbs2 mutant strain, in which the HOG pathway is blocked and Hog1p stays in the cytoplasm.

They found that fluorescence recovery took less than one second in normal media, about five seconds in 1 M sorbitol, and never happened at 2 M sorbitol. Clearly the protein’s movement was slowed with increasing osmotic pressure. But the effects weren’t permanent: once the cells were put back into normal media, the protein returned to its original kinetics.

The authors expanded their studies to look at proteins that shuttle between the nucleus and cytoplasm in other pathways, including Msn2p, Yap1p, Crz1p, and Mig1p, and obtained similar results. All four proteins took longer to reach the nucleus after being exposed to high osmotic stress. They also looked at other cellular processes where protein movement is critical, such as endocytosis, and again found that increased osmotic stress led to slower moving proteins.

So it looks like molecular crowding can definitely slow down protein movement and affect how a cell functions. For example, the time it takes to respond to environmental stimuli could be significantly slowed, affecting the cell’s chances for survival. Since yeast cells in the wild encounter high-sugar environments (like rotting grapes), their protein density must be regulated so that they can get through stresses like this. They are ultimately much better adapted than that goldfish in the bowl of Jell-O.